The present invention relates to microscopes and methods of obtaining images therewith. More particularly, the present invention relates to a method of obtaining images with Inverted Darkfield Contrast (IDC) microscopes and a novel IDC microscope.
For many years light microscopes have been considered a mature technology. While there have been notable attempts to extend the capabilities of the light microscope, to date such attempts have not achieved substantial gains in performance and have generally been obtained at significantly increased costs. Vibrations in microscopes have been known as a major factor contributing to the limit on resolving power. Vibrations in the microscope frame have previously been addressed by building super rigid or heavy frames, or by constructing horizontal microscopes on massive optical bench-style frames. Other attempts to improve the vibration performance have used passive or active vibration damping tables, feet or platforms.
The generation of image contrast in microscopes is an area where there has been considerable work carried out in the past. Attempts to increase the contrast of observed biological samples have resulted in many new methods such as phase contrast, interference contrast, Hoffman modulation contrast, differential interference contrast, polarized light microscopy, darkfield microscopy and fluorescent microscopy. The challenge of generating image contrast at the extreme limit of resolution yielded such techniques as high power immersion darkfield, and ultramicroscopic illumination. Phase and interference contrast techniques introduced artifacts, some of which were asymmetrical, which made the images difficult to relate to the real structure of the samples being viewed. Darkfield and fluorescent techniques presented image information in a form that is most unfamiliar to visual capabilities, much in the same way that we are unable to extract information from a photographic or electronic xe2x80x9cnegativexe2x80x9d image.
Attempts to gain more information about cells in real time has yielded confocal microscopy which uses high power laser light sources which scan the sample area to build a final image of the sample, and newer masked confocal techniques that can build higher speed images of live samples. In general, the frame/field rate of the confocal systems is too slow for studying the high speed motion of many components in biological systems since they exhibit high speed motion.
Attempts to yield high resolution have been based on the formula for microscopic resolution first developed by Ernst Abbe, resolution limit=wavelength of light/ (kxc3x97numerical aperture of the objective). Values for k ranging from 1.6 to 2 have been accepted for over 50 years but the inventor""s recent work suggests the value of k can be lowered and needs to be more fully studied when applied to improved optical systems with new methods of illumination and imaging means.
As microscope systems have become more complex, more glass surfaces created more light loss due to transmission losses in the glass elements, internal reflection and stray light. The stray light contributed to poor contrast and the internal reflections and transmission losses, together with the stray light, meant that progressively more powerful light sources were needed to produce useable image brightness. These high powered sources must propagate the light at high fluxes through the sample space since most of the lossy components are between the sample and the imaging means. Modern binocular and trinocular systems with their attendant prisms, mirrors and lenses are particularly inefficient and require higher light levels.
It is an object of the present invention to provide a novel IDC microscope and a novel method of obtaining images with an IDC.
According to a first aspect of the present invention, there is provided a novel method of achieving contrast for microscopical imaging of preparations of living cells and other types of objects is described along with improvements to microscopes. This method combines the traditional darkfield illumination technique with electronic image inversion (converting a positive to a negative image) and other improvements to further enhance the contrast and resolution of the final image. The method is referred to herein as Inverted Darkfield Contrast and is believed to be particularly suitable for viewing live cells in real time with no staining or preparation.
The embodiments shown herein are primarily based on a video microscope in which image resolution, contrast and optical efficiency are optimized. In microscopes in accordance with the present invention there is usually no intervening binocular or trinocular arrangement or eyepiece between the objective and the imaging system, which can be any type of imaging means including film cameras, analog or digital video cameras or image intensifiers. The microscope system can use a pre-focused and aligned lamp and reflector to direct a larger than usual portion of the light from the lamp into the illuminating beam. The illuminating beam is directed through a beam expander which controls the diameter of the illuminating beam while maintaining parallel rays of light. The illuminating beam passes through apertures to control stray light.
Careful attention is paid to controlling the illuminating wavelengths of light to improve the resolution of the microscope. In particular, all the non-visible wavelengths in the ultraviolet (UV) and infrared (IR) portion of the spectrum are preferably eliminated to improve image quality. The rays of light leaving the objective are also passed through apertures and baffle tube(s) to reduce stray light and enhance contrast. Anti-vibration means are also provided to control the motion of the objective, relative to the sample being viewed, and the position of the imaging device relative to the objective. Control of stray light in the objective and in the coupler between the objective and the imaging device also help to improve contrast and resolution.
The signal from the imaging device is inverted to form the negative of the normal image. In this way the traditional darkfield image appears as a high contrast brightfield image in the final monitor or computer display.
The present invention comprises a variety of mechanical and optical improvements to a microscope in order to achieve Inverted Darkfield Contrast (IDC). More specifically, a video microscope is provided which can include improvements to the illumination system, the condenser, the slide holder, the objectives, the tube, the microscope stand and the image acquiring system to produce a novel IDC microscope.
The present invention provides a method for obtaining high contrast images of living biological samples such as cells in real time with no staining or fluorochemistry required. The method is applicable to imaging a variety of materials, substances and structures, including cells, internal cellular structures, bacteria, viruses, fungi and plant materials. The present invention also includes improvements in microscope technology including improvements to stand design, illuminators, condensers, objectives, imaging systems and to video processing.
While the concept of darkfield imaging is not new and the use of video positive to negative inversion is known in the television broadcast special effects field, the present invention is the first application of these unrelated techniques to obtain high contrast images of samples such as living biological material. The present invention provides particular advantages as it can provide images which look like stained biological materials, so that biologists can readily interpret and accept the information that the images present, without requiring the staining of the imaged samples. The present invention can improve the contrast, resolution and speed of acquisition of the image, without significantly increasing the cost or complexity of the microscope.